Al-2 Quorum Sensing Regulation In Staphylococcus Aureus

Recently people found cell-cell communication is not just in multi-cellular organism, bacteria can also communicate with one another to coordinate their behavior. In the process of proliferation, bacteria secrete small diffusible molecules called autoinducers (AIs) continuously. While bacteria proliferated, AIs accumulated in the around environment, once reached a threshold concentration AIs can be detected and enter the cell, bind to its receptors and regulate transcription of lots of target genes. Thus bacteria can coordinate social behavior at multi-cellular level, such as bioluminescence, biofilm formation, swarming behavior, antibiotic production, and virulence factor secretion. This process is referred to as quorum sensing (QS), or density-dependent gene regulation, and this system is called QS system. Many QS mechanisms have evolved among bacteria. In general, gram-negative bacteria use acylated homoserine lactones (AHLs) as AIs, and gram-positive bacteria use oligopeptide AIs, which act through two-component phosphorelay cascades. There is one mechanism that is shared by both gram-positive and gram-negative bacteria, involving the production of autoinducer 2 (AI-2). In contrast to other autoinducers that are species specific, AI-2 is widely present in bacteria, leading to the suggestion that it is a universal language for interspecies communication. AI-2 is byproduct synthesized by LuxS enzyme in a metabolic pathway known as the activated methyl cycle. LuxS is conserved and widely present in both gram-negative and gram-positive bacteria. Due to its dual roles in metabolism and in QS, has been a hot topic to study, however, it is difficulty to find out its real rule in QS since it is not the main QS system and it is regulated by other regulators. The LuxS/AI-2 system is known to be functional as a QS system involved in the regulation of a range of behaviours in diverse bacteria, such as bioluminescence in Vibrio harveyi, biofilm formation in Escherichia coli, virulence-associated traits in Vibrio cholerae, and antibiotic susceptibility in Streptococcus anginosu. However, AI-2 QS role in gram-positive bacteria, especially in S. aureus, has not been studied in detail until now.S. aureus is a major nosocomial pathogen with the ability to cause a variety of infectious diseases, from relatively benign skin infections such as pimples, impetigo, furuncles, and scalded skin syndrome, to potentially fatal systemic disorders such as pneumonia, meningitis, osteomyelitis and septicemia. The strong pathogenesis of S. aureus is essentially determined by its virulence factors, including surface-associated adhesins, superantigens, exoenzymes and exotoxin, which are regulated by a wide range of regulatory systems. Among these regulatory elements, the Agr system (the accessory gene regulator) is the only characterized QS system in S. aureus. Interestingly, S. aureus also possesses a functional luxS gene and has the capability to produce AI-2. It will be of great importance to explore whether AI-2 can function as a QS signal to regulate physiological functions in S. aureus.In this study, we analyzed the transcript differences between the standard strains of S. aureus NCTC8325 wild type, the luxS mutant and the luxS mutant added exogenous AI-2 using microarray and real time RT-PCR. Our data show that Inactivation of luxS in S. aureus NCTC8325 resulted in higher transcription of capsular polysaccharide (CP) synthesis genes and the two component system kdpDE genes. The survival rate of the luxS mutant was higher than that of the wild type both in human blood and macrophage U937. Addition of exogenous AI-2 restored all the parental phenotypes by a concentration-dependent mechanism. CP is an important cell wall component that can interact with the host immune system during the invasive process, allowing the organism to resist uptake and killing by phagocytes. It is interesting that the transcript levels of various regulatory elements known to modulate CP synthesis in S. aureus such as agrA, sarA, sbcDC, rnaâ…¢arlRS, ccpA, mgrA, saeRS, and spoVG displayed no apparent changes in luxS mutant. So we suspected that LuxS/AI-2 modulated cap gene transcription through another mechanism. Sensor protein KdpD together with reaction protein KdpE constitutes a two-component signal transduction system, which was first characterized in E. coli. In this organism, KdpDE regulate severe K+ limitation or osmotic upshift. In Mycobacterium tuberculosis, deletion of kdpDE resulted in increased virulence. Although several reports have shown that, in S. aureus, the transcript level of kdpDE changes under certain environmental stresses, information about the role of KdpDE in S. aureus and how it functions remains incomplete.To study the relationship between KdpDE and CP, we constructed kdpDE mutant strain. Our data show that the transcript levels of cap genes were decreased in kdpDE mutant and gel-shift assay show that KdpE can bind to the promoter region of the cap operon, suggesting that the KdpDE system could regulate cap gene transcription. kdpE mutant and KdpE phosphorylation site mutant assays indicated that KdpE regulates the transcription of cap through a phosphorylation pathway. The alterated survival of S. aureus in human blood and monocytic cells correlated with the changes in transcript levels of CP. The data further validated our hypothesis that S. aureus AI-2 quorum sensing regulates CP synthesis and virulence through the two-component regulatory system KdpDE. Our study provided a direct proof that the LuxS/AI-2 system played a signaling role in S. aureus and new clues to the functional analysis of the KdpDE system in S. aureus. Our findings add new understanding to AI-2 quorum sensing regulation and mechanisms of innate immune evasion used by S. aureus and provide novel clues for antimicrobial chemotherapy of Staphylococal infection.